EPF-Lausanne/6 July 2009

From 2009.igem.org

Contents

6 July 2009





Wet Lab

LOVTAP plasmid AND TrpR plasmid were transformed in competent E. Coli following received protocol, and grown overnight (see Lab book for more details).
One problem: we actually don't know TrpR plasmid resistance, so we tried with three resistances available in the lab: Amp., Kana. and Chl. LOVTAP is in a plasmid called pCal-n (see picture below):

pCal-n plasmid


Some comments on the plasmid:
-CBP is a small peptide with which we could purify LOVTAP protein
-Thrombin target is a nucleotidic sequence that can be recognized by thrombin a peptidase. This peptidase will cut CBP once LOVTAP is purified


Cloning Strategy

Four forward primers were designed to amplify:
1.Promoter T7, RBS, CBP and LOVTAP:

gtttcttcgaattcgcggccgcttctagagtaatacgactcactataggggaattgtg

2.RBS, CBP and LOVTAP:

gtttcttcgaattcgcggccgcttctagagtgtttaactttaagaaggag

3.CBP and LOVTAP:

gtttcttcgaattcgcggccgcttctagatgaagcgacgatggaaaaagaatttcatag

4.LOVTAP:

gtttcttcgaattcgcggccgcttctagatgctactacacttgaacgtattgagaagaac

One reverse primer were designed:

gtttcttcctgcagcggccgctactagtatcaatcgcttttcagcaacacctcttc

The recipient IGEM part have been chosen: BBa_B0010, well 13D in the received kit plate 1


People in the lab

Tu, Heidi, Rafael, Basile, Nath